Glycoprotein B of herpes simplex virus 2 has more than one intracellular conformation and is altered by low pH

The crystal structure of herpes simplex virus (HSV) gB identifies it as a class III fusion protein, and comparison with other such proteins suggests this is the postfusion rather than prefusion conformation, although this is not proven. Other class III proteins undergo a pH-dependent switch between pre- and postfusion conformations, and a low pH requirement for HSV entry into some cell types suggests that this may also be true for gB. Both gB and gH undergo structural changes at low pH, but there is debate about the extent and significance of the changes in gB, possibly due to the use of different soluble forms of the protein and different assays for antigenic changes. In this study, a complementary approach was taken, examining the conformations of full-length intracellular gB by quantitative confocal microscopy with a panel of 26 antibodies. Three conformations were distinguished, and low pH was found to be a major influence. Comparison with previous studies indicates that the intracellular conformation in low-pH environments may be the same as that of the soluble form known as s-gB at low pH. Interestingly, the antibodies whose binding was most affected by low pH both have neutralizing activity and consequently must block either the function of a neutral pH conformation or its switch from an inactive form to an activated form. If one of the intracellular conformations is the fusion-active form, another factor required for fusion is presumably absent from wherever that conformation is present in infected cells so that inappropriate fusion is avoided.     

Residues within the C-terminal arm of the herpes simplex virus 1 glycoprotein B ectodomain contribute to its refolding during the fusion step of virus entry

Herpesvirus entry into cells requires coordinated interactions among several viral glycoproteins. The final membrane fusion step of entry is executed by glycoprotein B (gB), a class III viral fusion protein that is conserved across all herpesviruses. Fusion proteins are metastable proteins that mediate fusion by inserting into a target membrane and refolding from a prefusion to postfusion conformation to bring the viral and cell membranes together. Although the structure of gB has been solved in a conformation that likely represents its postfusion form, its prefusion structure and the details of how it refolds to execute fusion are unknown. The postfusion gB structure contains a trimeric coiled-coil at its core and a long C-terminal arm within the ectodomain packs against this coil in an antiparallel manner. This coil-arm complex is reminiscent of the six-helix bundle that provides the energy for fusion in class I fusogens. To determine the role of the coil-arm complex, we individually mutated residues in the herpes simplex virus 1 gB coil-arm complex to alanine and assessed the contribution of each residue to cell-cell and virus-cell fusion. Several coil mutations resulted in a loss of cell surface expression, indicating that the coil residues are important for proper processing of gB. Three mutations in the arm region (I671A, H681A, and F683A) reduced fusion without affecting expression. Combining these three arm mutations drastically reduced the ability of gB to execute fusion; however, fusion function could be restored by adding known hyperfusogenic mutations to the arm mutant. We propose that the formation of the coil-arm complex drives the gB transition to a postfusion conformation and the coil-arm complex performs a function similar to that of the six-helix bundle in class I fusion. Furthermore, we suggest that these specific mutations in the arm may energetically favor the prefusion state of gB.     

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion

Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.     

Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.     

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.     

Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation

Glycoprotein B (gB) enables the fusion of viral and cell membranes during entry of herpesviruses. However, gB alone is insufficient for membrane fusion; the gH/gL heterodimer is also required. The crystal structure of the herpes simplex virus type 1 (HSV-1) gB ectodomain, gB730, has demonstrated similarities between gB and other viral fusion proteins, leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the membranes together by refolding from its initial or prefusion form to its final or postfusion form. The only available crystal structure likely represents the postfusion form of gB; the prefusion form has not yet been determined. Previously, a panel of HSV-1 gB mutants was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite being expressed on the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions might not be accommodated in the postfusion form. Thus, we hypothesized that some insertion mutants were nonfunctional due to being “trapped” in a prefusion form. Here, we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We show that the ectodomains of all five mutants assume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall structures that are indistinguishable from those of the wild-type gB730. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, one mutant, while able to adopt the overall postfusion structure, displays significant conformational differences in the vicinity of fusion loops, relative to wild-type gB730. Moreover, this mutant has a diminished ability to associate with liposomes, suggesting that the fusion loops in this mutant have decreased functional activity. We propose that these insertions cause a fusion-deficient phenotype not by preventing conversion of gB to a postfusion-like conformation but rather by interfering with other gB functions.Herpes simplex virus type 1 (HSV-1) is the prototype of the diverse herpesvirus family that includes many notable human pathogens (26). In addition to the icosahedral capsid and the tegument that surround its double-stranded DNA genome, herpesviruses have an envelope—an outer lipid bilayer—bearing a number of surface glycoproteins. During infection, HSV-1 must fuse its envelope with a cellular membrane in order to deliver the capsid into a target host cell. Among its viral glycoproteins, only glycoprotein C (gC), gB, gD, gH, and gL participate in this entry process, and only the last four are required for fusion (28). Although gD is found only in alphaherpesviruses, all herpesviruses encode gB, gH, and gL, which constitute their core fusion machinery. Of these three proteins, gB is the most highly conserved.We recently determined the crystal structure of a nearly full-length ectodomain of HSV-1 gB, gB730 (18). The crystal structure of the ectodomain of gB from Epstein-Barr virus, another herpesvirus, has also been subsequently determined (4). The two structures showed similarities between gB and other viral fusion proteins, in particular, G from an unrelated vesicular stomatitis virus (VSV) (25), leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the viral and host cell membranes together to enable their fusion. However, gB alone is known to be insufficient for membrane fusion; the gH/gL heterodimer is also required. This insufficiency raises the question of exactly how gB functions during viral entry. Answering this question is critical for understanding the complex mechanism that herpesviruses use to enter their host cells.In acting as a viral fusogen, gB must undergo dramatic conformational changes, refolding through a series of conformational intermediates from its initial, or prefusion form, to its final, or postfusion form (15). These conformational changes are not only necessary to bring the two membranes into proximity; they are also thought to provide the energy for the fusion process. The prefusion form corresponds to the protein present on the viral surface prior to initiation of fusion. The postfusion form represents the protein after fusion of the viral and host cell membranes. The available gB structure likely represents its postfusion form, since it shares more in common with the postfusion rather than the prefusion structure of vesicular stomatitis virus (VSV) G (3, 17). However, the prefusion form has not yet been characterized.Recently, a panel of gB mutants was generated by using random linker-insertion mutagenesis (21). Of these mutants, 16 were particularly interesting because they were nonfunctional in cell-cell fusion assays despite being expressed on the cell surface at levels that indicate proper folding for transport. These observations suggested that each insertion somehow interfered with gB function. Insertions in 12 of these mutants are located within the available structure of the gB ectodomain, which allowed Lin and Spear to analyze their locations (21).The most prominent examples of such nonfunctional mutants are two mutants with insertions after residues I185 or E187, henceforth referred to as “cavity mutants” because both I185 and E187 point into a cavity inside the gB trimer (Fig. 1B and D). Although this cavity might accommodate a single 5-amino-acid insertion, it “is not large enough to accommodate three 5-amino-acid insertions” (21) that would be present in the trimer (one insertion per protomer).Open in a separate windowFIG. 1.Location of the insertion sites in the sequence of gB and the structure of the postfusion form of its ectodomain. (A) Linear diagram of the full-length gB with functional domains highlighted (as in reference 18). Domain I is shown in cyan, domain II in green, domain III in yellow, domain IV in orange, domain V in red, and the disordered region between domains II and III in purple. Regions absent from the crystal structure of gB730 are shown in gray. Sequences in the region of 5-amino-acid insertions (residues 181 to 190 and residues 661 to 680) are shown in black. Arrows mark the locations of 5-amino-acid insertions, shown as red text. (B) Crystal structure of gB730 (18). Residues preceding the 5-amino-acid insertions in mutants studied here are shown as spheres colored by domain, consistent with panel A. Boxes delineate the hinge region, enlarged in panel C, and the cavity region, enlarged in panel D. (C) Close-up view of the hinge region shown in molecular surface representation, with residues 663 to 675 displayed as sticks. Hydrophobic residues are colored orange. Residues preceding the 5-amino-acid insertions in mutants studied here are labeled with asterisks; remaining labels correspond to additional hydrophobic residues in the 663-675 region. (D) Enlarged view of the cavity region. Residues that line the cavity and are not solvent exposed are colored magenta. Residue E187 of each protomer is colored teal and shown as spheres. Fusion loops for two protomers are marked with asterisks; the third pair of fusion loops lies behind the crystal structure and is not visible. Panels B, C, and D were made by using Pymol (http://www.pymol.org/).Five other nonfunctional mutants have insertions after residues D663, T665, V667, I671, or L673, respectively. We refer to them as “hinge mutants.” These residues lie in the region located between domains IV and V, which has been termed the hinge region because it may play an important role during the conformational transition from the prefusion to the postfusion form (17). Lin and Spear proposed that insertions following these residues “would likely affect hinge regions” (21), with the implication that they may prevent gB from refolding into the postfusion conformation. Our analysis suggested that insertions after these residues could, perhaps, be sterically accommodated in the structure but would probably be energetically unfavorable by causing several buried hydrophobic side chains in the 665-673 region, such as F670, I671, and L673, to become exposed (Fig. 1B and C).In light of these observations, we hypothesized that the insertion mutants are “trapped” in a prefusion form. We decided to test this hypothesis by determining whether the ectodomain of gB containing one of these insertion mutations is able to assume the conformation seen in the crystal structure of the wild-type gB ectodomain, which we are referring to as the likely postfusion conformation. For this purpose, we chose one cavity mutant, containing an insertion after E187, and four hinge mutants, containing insertions after T665, V667, I671, or L673, respectively. We chose to test four hinge mutants because structure analysis suggested to us that insertions following the respective residues might not affect the structure in precisely the same way. We expressed the soluble ectodomain of each mutant by using a baculovirus expression system and characterized the purified proteins by using biochemical and biophysical methods. Surprisingly, we found that the ectodomains of all five mutants assume a conformation similar to that of the wild-type gB ectodomain. The four hinge mutants had biochemical properties and overall three-dimensional structures that were indistinguishable from those of the wild-type gB ectodomain. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, the cavity mutant, while able to adopt the overall postfusion structure, still displayed significant conformational differences relative to wild-type gB. Because these conformational differences are in the vicinity of fusion loops, we conclude that the fusion loops in this mutant have decreased functional activity.     

Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.Herpes simplex virus (HSV) is an important human pathogen, causing significant morbidity and mortality worldwide. HSV enters host cells by fusion of the viral envelope with either an endosomal membrane (38) or the plasma membrane (63). The entry pathway taken is thought to be determined by both virus (17, 45) and host cell (4, 17, 35, 39, 45) factors. Based on experiments with lysosomotropic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytic entry of HSV into several cell types, including human epithelial cells (37). Low pH has also recently been implicated in cell infection by several other human and veterinary herpesviruses (1, 21, 26, 47). The mechanistic role of endosomal pH in herpesvirus entry into cells is not known.Herpesviruses are a paradigm for membrane fusion mediated by a complex of several glycoproteins. We have proposed that HSV likely encodes machinery to mediate both pH-dependent and pH-independent membrane fusion reactions. Envelope glycoproteins glycoprotein B (gB) and gD and the heterodimer gH-gL are required for both pH-independent and pH-dependent entry pathways (11, 22, 30, 39, 46). Interaction of gD with one of its cognate receptors is an essential trigger for membrane fusion and entry (13, 52), regardless of the cellular pathway. However, engagement of a gD receptor is not sufficient for fusion, and at least one additional unknown trigger involving gB or gH-gL is likely necessary. gB is conserved among all herpesviruses, and in all cases studied to date, it plays roles in viral entry, including receptor binding and membrane fusion. The crystal structure of an ectodomain fragment of HSV type 1 (HSV-1) gB is an elongated, rod-like structure containing hydrophobic internal fusion loops (28). This structure bears striking architectural homology to the low pH, postfusion form of G glycoprotein from vesicular stomatitis virus (VSV-G) (43). Both the gB and G structures have features of class I and class II fusion proteins and are thus designated class III proteins (57).During entry of the majority of virus families, low pH acts directly on glycoproteins to induce membrane fusion (60). In some cases, the low pH trigger is not sufficient, or it plays an indirect role. For example, host cell proteases, such as cathepsins D and L, require intravesicular low pH to cleave Ebola virus and severe acute respiratory syndrome (SARS) glycoproteins to trigger fusion (14, 51).We investigated the role of low pH in the molecular mechanism of herpesviral entry. The results suggest that mildly acidic pH, similar to that found within endosomes, triggers a conformational change in gB. We propose that, together with other cellular cues such as receptor interaction, intracellular low pH can play a direct activating role in HSV membrane fusion and entry.     

Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV-1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pH-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the glycoproteins. Infection of cells expressing gB or gD glycoproteins with HSV-1 inhibited the low pH-induced cell fusion. The results suggest that although the glycoproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.     

Capturing the herpes simplex virus core fusion complex (gB-gH/gL) in an acidic environment

Herpes simplex virus (HSV) entry requires the core fusion machinery of gH/gL and gB as well as gD and a gD receptor. When gD binds receptor, it undergoes conformational changes that presumably activate gH/gL, which then activates gB to carry out fusion. gB is a class III viral fusion protein, while gH/gL does not resemble any known viral fusion protein. One hallmark of fusion proteins is their ability to bind lipid membranes. We previously used a liposome coflotation assay to show that truncated soluble gB, but not gH/gL or gD, can associate with liposomes at neutral pH. Here, we show that gH/gL cofloats with liposomes but only when it is incubated with gB at pH 5. When gB mutants with single amino acid changes in the fusion loops (known to inhibit the binding of soluble gB to liposomes) were mixed with gH/gL and liposomes at pH 5, gH/gL failed to cofloat with liposomes. These data suggest that gH/gL does not directly associate with liposomes but instead binds to gB, which then binds to liposomes via its fusion loops. Using monoclonal antibodies, we found that many gH and gL epitopes were altered by low pH, whereas the effect on gB epitopes was more limited. Our liposome data support the concept that low pH triggers conformational changes to both proteins that allow gH/gL to physically interact with gB.     

Structural characterization of an early fusion intermediate of influenza virus hemagglutinin

The hemagglutinin (HA) envelope protein of influenza virus mediates viral entry through membrane fusion in the acidic environment of the endosome. Crystal structures of HA in pre- and postfusion states have laid the foundation for proposals for a general fusion mechanism for viral envelope proteins. The large-scale conformational rearrangement of HA at low pH is triggered by a loop-to-helix transition of an interhelical loop (B loop) within the fusion domain and is often referred to as the "spring-loaded" mechanism. Although the receptor-binding HA1 subunit is believed to act as a "clamp" to keep the B loop in its metastable prefusion state at neutral pH, the "pH sensors" that are responsible for the clamp release and the ensuing structural transitions have remained elusive. Here we identify a mutation in the HA2 fusion domain from the influenza virus H2 subtype that stabilizes the HA trimer in a prefusion-like state at and below fusogenic pH. Crystal structures of this putative early intermediate state reveal reorganization of ionic interactions at the HA1-HA2 interface at acidic pH and deformation of the HA1 membrane-distal domain. Along with neutralization of glutamate residues on the B loop, these changes cause a rotation of the B loop and solvent exposure of conserved phenylalanines, which are key residues at the trimer interface of the postfusion structure. Thus, our study reveals the possible initial structural event that leads to release of the B loop from its prefusion conformation, which is aided by unexpected structural changes within the membrane-distal HA1 domain at low pH.     

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary,Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.     

Structural intermediates in the low pH-induced transition of influenza hemagglutinin

The hemagglutinin (HA) glycoproteins of influenza viruses play a key role in binding host cell receptors and in mediating virus-host cell membrane fusion during virus infection. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. While structures of prefusion state and postfusion state of HA have been reported, the intermediate structures remain elusive. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). Our results show that a decrease in pH from 7.8 to 5.2 triggers the release of fusion peptides from the binding pockets and then causes a dramatic conformational change in the central helices, in which the membrane-proximal ends of the central helices unwind to an extended form. Accompanying the conformational changes of the central helices, the stem region of the HA undergoes an anticlockwise rotation of 9.5 degrees and a shift of 15 Å. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. Thus, the conformational change of the HA stem region observed in our research is likely to be independent of the HA head. These results provide new insights into the structural transition of HA during virus entry.     

Viral fusion mechanisms

The majority of viral fusion proteins can be divided into two classes. The influenza hemagglutinin (HA) belongs to the class I fusion proteins and undergoes a series of conformational changes at acidic pH, leading to membrane fusion. The crystal structures of the prefusion and the postfusion forms of HA have been revealed in 1981 and 1994, respectively. On the basis of these structures, a model for the mechanism of membrane fusion mediated by the conformational changes of HA has been proposed. The flavivirus E and alphavirus E1 proteins belong to the class II fusion proteins and mediate membrane fusion at acidic pH. Their prefusion structures are distinct from that of HA. Last year, however, it has become evident that the postfusion structures of these class I and class II fusion proteins are similar. The paramyxovirus F protein belongs to the class I fusion proteins. In contrast to HA, an interaction between F and its homologous attachment protein is required for F to undergo the conformational changes. Since F mediates fusion at neutral pH, the infected cells can fuse with neighboring uninfected cells. The crystal structures of F and the attachment protein HN have recently been clarified, which will facilitate studies of the molecular mechanism of F-mediated membrane fusion.     

Autographa californica multiple nucleopolyhedrovirus GP64 protein: roles of histidine residues in triggering membrane fusion and fusion pore expansion

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein mediates membrane fusion during entry. Fusion results from a low-pH-triggered conformational change in GP64 and subsequent interactions with the membrane bilayers. The low-pH sensor and trigger of the conformational change are not known, but histidine residues are implicated because the pK(a) of histidine is near the threshold for triggering fusion by GP64. We used alanine substitutions to examine the roles of all individual and selected clusters of GP64 histidine residues in triggering and mediating fusion by GP64. Three histidine residues (H152, H155, and H156), located in fusion loop 2, were identified as important for membrane fusion. These three histidine residues were important for efficient pore expansion but were not required for the pH-triggered conformational change. In contrast, a cluster of three histidine residues (H245, H304, and H430) located near the base of the central coiled coil was identified as a putative sensor for low pH. Three alanine substitutions in cluster H245/H304/H430 resulted in dramatically reduced membrane fusion and the apparent loss of the prefusion conformation at neutral pH. Thus, the H245/H304/H430 cluster of histidines may function or participate as a pH sensor by stabilizing the prefusion structure of GP64.     

The Roles of Histidines and Charged Residues as Potential Triggers of a Conformational Change in the Fusion Loop of Ebola Virus Glycoprotein

Ebola virus (EBOV) enters cells from late endosomes/lysosomes under mildly acidic conditions. Entry by fusion with the endosomal membrane requires the fusion loop (FL, residues 507–560) of the EBOV surface glycoprotein to undergo a pH-dependent conformational change. To find the pH trigger for this reaction we mutated multiple conserved histidines and charged and uncharged hydrophilic residues in the FL and measured their activity by liposome fusion and cell entry of virus-like particles. The FL location in the membrane was assessed by NMR using soluble and lipid-bound paramagnetic relaxation agents. While we could not identify a single residue to be alone responsible for pH triggering, we propose that a distributed pH effect over multiple residues induces the conformational change that enhances membrane insertion and triggers the fusion activity of the EBOV FL.     

Impact of valency of a glycoprotein B-specific monoclonal antibody on neutralization of herpes simplex virus

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab')(2)] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.     

Hepatitis C virus is primed by CD81 protein for low pH-dependent fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.     

Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B,Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics

The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.     

Characterization of a structural intermediate of flavivirus membrane fusion

Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E) mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion.     

On the Stability of Parainfluenza Virus 5 F Proteins

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.